Thermostable engineered enzyme

Abstract

The invention relates to an engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1. Preferably, the PETase enzyme comprises SEQ ID NO.1 with at least four mutations. The invention also refers to a composition and to a method for decomposing plastics.

Description

[0001] Thermostable engineered enzyme

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a novel plastic degrading enzyme and a plastic decomposition method using the same.

[0004] STATE OF THE ART

[0005] PET (Polyethylene terephthalate) is a type of thermoplastic made by polymerization of TPA (terephthalic acid) and EG (ethylene glycol). PET has high transparency and excellent thermal insulation properties, so it is a polymer material commonly used in electric wire coverings, household goods, toys, and packaging materials, and is particularly widely used in the manufacture of bottles. However, since PET has a chemical structure that is not easily decomposed in nature, environmental pollution caused by the accumulation of PET in ecosystems including soil, rivers, and oceans is recognized as a serious social problem. In particular, the bioaccumulation problem caused by microplastics can adversely affect human health, so the need for recycling or environmentally friendly decomposition of PET is gradually increasing.

[0006] However, when PET is recycled industrially, the recycling rate is not high due to poor quality and high cost, and when PET is decomposed through physical and chemical methods, endocrine-disrupting chemicals such as dioxins can be produced, which is a secondary problem. There is a problem that occurs.

[0007] In 1977, Tokiwa and Suzuki proposed the idea of using enzymes to degrade polymeric materials. Indeed, enzymes work in mild conditions and can replace hazardous chemicals (a concept known as green chemistry). Since then, many PET-degrading enzymes from various microorganisms have been discovered and characterized, which has also led to the implementation of a series of strategies for enhancing their catalytic properties. Despite the availability of many suitable enzymes, their practical use for PET degradation is limited, mostly due to their low thermal stability. Indeed, the enzymatic degradation of PET by PETases does improve at high temperatures, where PET crystallinity is reduced. Hence, the design of a PETase with enhanced thermal stability is particularly relevant to the development of enzymatic PET-degrading strategies.

[0008] SUMMARY OF THE INVENTION

[0009] The first aspect of the invention relates to an engineered PETase enzyme. In one embodiment, the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1 .

[0010] Preferably, the PETase enzyme comprising SEQ ID NO.1 with at least four mutations, also referred as amino acid substitutions. In one embodiment, said SEQ ID NO.1 comprises at least four mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.

[0011] A second aspect of the present invention refers to a composition for decomposing plastics.

[0012] The composition for decomposing plastic comprises the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 2 and / or the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 3.

[0013] Another aspect of the present invention provides a method for decomposing plastics.

[0014] The method for decomposing plastics includes treating the plastics with the composition for decomposing plastics of the present invention. The plastic may be MHET, BHET, PET, or the like, or derivatives thereof.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] The Figures show a comparison of PET-depolymerization over time for C09, C08 and ICCG (40 nM, 80 °C, pH 8). Means ± s.d (n=2) are shown. Figure 1 shows TPA production over time;

[0017] Figure 2 shows TPA production at 4h and 144h. *P<0.05; **P<0.01 (one- side unpaired Welch’s t-test); and

[0018] Figure 3 shows enzyme specific activity at the different time points.

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0020] The first aspect of the invention relates to an engineered PETase enzyme. The term “PETase” or “polyethylene terephthalate hydrolases” refer to a class of hydrolases that have the property of catalysing the cleavage of PET (polyethylene terephthalate) into mono (2-hydroxyethyl) terephthalic acid.

[0021] In one embodiment, there is provided an engineered thermostable PETase enzyme with a melting temperature (Tm) comprised between 80°C and 100°C, preferably, comprised between 82 and 98°C, that is higher than wild type enzyme. The engineered PETase enzyme may be active at higher temperatures (80-90 °C) enabling more efficient degradation of substrates. The thermostable PETase enzyme may also have longer shelf life at ambient temperatures.

[0022] In one embodiment, the PETase enzyme has improved thermal stability. In one embodiment, the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 1 .

[0023] Preferably, the PETase enzyme comprises SEQ ID NO.1 with at least four mutations, also referred as amino acid substitutions. In one embodiment, said SEQ ID NO.1 comprises at least four mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K. In other words, the enzyme comprises SEQ ID NO. 1 with at least four mutations in the above-identified positions.

[0024] In some embodiments, SEQ ID NO.1 comprises at least five mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K. Preferably, SEQ ID NO.1 comprises at least six mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.

[0025] In some embodiments, SEQ ID NO.1 comprises at least seven mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K. Preferably, SEQ ID NO. 1 comprises the mutations: S48D, S57K, S145R, A209R, S241 D, N276D, N278K, Q293K.

[0026] In one embodiment, the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 2.

[0027] In a preferred embodiment, the PETase enzyme consists of SEQ ID NO.2. Preferably, SEQ ID NO. 1 comprises the mutations: S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K, Q293K.

[0028] In one embodiment, the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 3.

[0029] In a preferred embodiment, the PETase enzyme consists of SEQ ID NO.3.

[0030]

[0031] Another aspect of the present invention provides a composition for decomposing plastics. The composition for decomposing plastic comprises at least one PTease enzyme as detailed disclosed above. Preferably, the composition comprises the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 2 and / or the engineered PETase enzyme comprises an amino acid sequence having at least 70% or 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 3.

[0032] In a preferred embodiment, the composition comprises the engineered PETase enzyme consisting of SEQ ID NO: 2 and / or the engineered PETase enzyme consisting of SEQ ID NO: 3. The plastic may be Mono-(2-hydroxyethyl)terephthalic acid (MHET) and Bis-(2-hydroxyethyl)terephthalic acid (BHET), and PET, or the like, or a derivative thereof. In some embodiments, the composition for decomposing plastics of the present invention comprises at least one ion selected from the group consisting of Ni2+, Fe2+, Co2+, Cu2+, Mg2+, Mn2+, K+, Zn2+ and EDTA, preferably at a concentration of 10 mM or less in the composition of the present invention.

[0033] Another aspect of the present invention provides a method for decomposing plastics.

[0034] The method for decomposing plastics includes treating the plastics with the enzyme and / or with the composition for decomposing plastics of the present invention.

[0035] Preferably, the plastic is selected from: MHET, BHET, PET, or the like, or derivatives thereof.

[0036] The step of treating the composition on the plastic may be carried out under the conditions of pH 7.5 to pH 10.5. Specifically, the pH may be in the range of pH 7.0 to pH 10.5, 7.5 to pH 10.0, or 7.5 to pH 9.0. The plastic degrading enzyme of the present invention can exhibit a higher activity in the above pH range, thereby exhibiting an activity of 80% or more of the maximum activity.

[0037] Preferably, the enzyme is used in a concentration comprised between 20 and 60 nM, more preferably comprised between 30 and 50 nM.

[0038] Example

[0039] Enzyme stability

[0040] To this end, the Applicant focused on the so-called leaf-branch compost cutinase (LCC), a naturally occurring PETase that has been reported to outperform all other known PET-degrading enzymes and presents a melting temperature (Tm) of 84.7°C. This enzyme has been previously engineered by Tournier et al. (Nature, 2020), leading to the ICCG variant with a reported Tm of 94.5°C (88.9°C in our own assessment). Starting from this ICCG variant, different enzyme design strategies have been applied to engineer new enzyme mutants. This led to two variants of the enzyme (named C08 and C09), one of which show a Tm higher than any other PETase currently known.

[0041] Enzyme Melting temperature [Tm, °C] Improvement

[0042] LCC (Wild type) 84.7

[0043] ICCG (Tournier et al.) 94.5 (reported)* +9.8*

[0044] 88.9 (our assessment)* +4.2*

[0045] C08 (this work) 82.2

[0046] C09 (this work) 96.8 +12.1

[0047] *The estimation of Tm is sensitive to the experimental setup and conditions, which could explain the difference between the reported Tm and the Tm determined by us. For proper comparison, we assessed the Tm of ICCG using the same setup that we used for the measurement of the Tm of our engineered enzymes.

[0048] Enzyme activity

[0049] Terephthalic acid (TPA) is one of the major degradation product of the activity of the enzymes on PET. This compound can be detected by bulk UV spectrophotometry allowing a precise quantification of product formation. Sample were harvested at multiple time points and analysed by ultra-high-performance liquid chromatography UHPLC for the quantification of TPA production. An enzyme concentration of 40 nM has been used to evaluate the PET degradation activity of the different mutants. The experiments were carried at 80°C and were monitored for 6 days (144h). For the selected mutants (i.e. , C09 and C08) the mean value of TPA concentration at the different time points is significantly higher for the mutated enzymes than for ICCG (Figure 1 and 2). In particular, the specific activity of C09 is much higher than the ICCG gold standard and, although it degrades over time, the mutant retains higher activity up until day 6 (144h, Figure 3).

Claims

CLAIMS1. An engineered PETase enzyme comprising SEQ ID NO.1 or a composition comprising it, wherein SEQ ID NO. 1 comprises at least four mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.

2. The enzyme according to claim 1 , wherein SEQ ID NO.1 comprises at least five mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.

3. The enzyme according to claim 1 or 2, wherein SEQ ID NO.1 comprises at least six mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.

4. The enzyme according to anyone of claims 1-3, wherein SEQ ID NO.1 comprises at least seven mutations selected from: S241 D, S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K and Q293K.

5. The enzyme according to anyone of claims 1-4, wherein SEQ ID NO. 1 comprises the mutations: S48D, S57K, S145R, A209R, S241 D, N276D, N278K, Q293K.

6. The enzyme according to claim 5, consisting of SEQ ID NO.2.

7. The enzyme according to anyone of claims 1-4, wherein SEQ ID NO. 1 comprises the mutations: S36D, Q40R, S48D, S57K, S145R, A209R, N276D, N278K, Q293K.

8. The enzyme according to claim 7 consisting of SEQ ID NO.3.

9. A method for decomposing plastics comprises at least one step oftreating the plastics with the enzyme and / or with the composition according to anyone of claims 1-8.

10. The method according to claim 9, wherein the plastic is selected from: MHET, BHET, PET, preferably is PET.11 . The method according to claim 9, or 10 wherein the enzyme is used in a concentration comprised between 20 and 60 nM, preferably comprised between 30 and 50 nM.

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